Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis

Karmakar, Shilpi; Ismail, Nevien; Oliveira, Fabiano; Oristian, James; Zhang, Wen-Wei; Kaviraj, Swarnendu; Singh, Kamaleshwar P.; Mondal, Abhishek; Das, Sushmita; Pandey, Krishna; Bhattacharya, Pinaki; Volpedo, Greta; Gannavaram, Sreenivas; Satoskar, Monika; Satoskar, Sanika; Sastry, Rajiv M; Oljuskin, Timur; Sepahpour, Telly; Meneses, Claudio; Hamano, Shinjiro; Das, Pradeep; Matlashewski, Greg; Singh, Sanjay; Kamhawi, Shaden; Dey, Ranadhir; Valenzuela, Jesús G.; Satoskar, Abhay R.; Nakhasi, Hira L.

doi:10.1038/s42003-021-02446-x

Cited by 37 publications

(59 citation statements)

References 49 publications

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“…Importantly, immunization with centrin −/− L. donovani provided strong protection against challenge with live parasites and immunized mice developed a multifunctional T cell response accompanied by a significant reduction in parasite load ( Selvapandiyan et al, 2009 ). Recently, these findings have been expanded to show that immunization with centrin-deficient L. major confers protection against infected sand flies ( Zhang et al, 2020b ; Karmakar et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing

Sharma

Avendaño-Rangel

Reis-Cunha

et al. 2022

Front. Cell. Infect. Microbiol.

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Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more accessible, and here we have successfully employed the LeishGEdit approach to attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a calcium-binding cytoskeletal protein involved in the centrosome duplication in eukaryotes. Centrin-deficient Leishmania exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen−/−) did not indicate the presence of off-target mutations. In vitro, the growth rates of LbCen−/− and wild-type promastigotes were similar, but axenic and intracellular LbCen−/− amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, LbCen−/− were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type L. braziliensis. A significantly lower parasite burden was also observed in mice inoculated with LbCen−/−, differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that LbCen−/− can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis.

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Section: Introductionmentioning

confidence: 99%

Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing

Sharma

Avendaño-Rangel

Reis-Cunha

et al. 2022

Front. Cell. Infect. Microbiol.

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“…This reserves selectable markers for additional genetic manipulation steps such as complementation. Currently, a marker-free approach has been used to attenuate Leishmania major for vaccine development (Zhang et al, 2020), a requirement for use in preclinical and clinical studies (Karmakar et al, 2021). The TcTrypanin −/− parasite we have developed here are suitable to be tested as a vaccine candidate, since it showed reduced motility and lower infectivity in vitro.…”

Section: Discussionmentioning

confidence: 99%

Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi

Saenz-Garcia

Borges

Souza-Melo

et al. 2022

Front. Cell. Infect. Microbiol.

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The flagellum of Trypanosomatids is an organelle that contributes to multiple functions, including motility, cell division, and host–pathogen interaction. Trypanin was first described in Trypanosoma brucei and is part of the dynein regulatory complex. TbTrypanin knockdown parasites showed motility defects in procyclic forms; however, silencing in bloodstream forms was lethal. Since TbTrypanin mutants show drastic phenotypic changes in mammalian stages, we decided to evaluate if the Trypanosoma cruzi ortholog plays a similar role by using the CRISPR-Cas9 system to generate null mutants. A ribonucleoprotein complex of SaCas9 and sgRNA plus donor oligonucleotide were used to edit both alleles of TcTrypanin without any selectable marker. TcTrypanin −/− epimastigotes showed a lower growth rate, partially detached flagella, normal numbers of nuclei and kinetoplasts, and motility defects such as reduced displacement and speed and increased tumbling propensity. The epimastigote mutant also showed decreased efficiency of in-vitro metacyclogenesis. Mutant parasites were able to complete the entire life cycle in vitro; however, they showed a reduction in their infection capacity compared with WT and addback cultures. Our data show that T. cruzi life cycle stages have differing sensitivities to TcTrypanin deletion. In conclusion, additional work is needed to dissect the motility components of T. cruzi and to identify essential molecules for mammalian stages.

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“…). LmCen -/parasites showed limited survival in the host and induced protective immunity comparable to leishmanization against both needle and sand fly challenge with a wildtype L. major as well as L. donovani infection 8,9 . We also demonstrated that upon challenge with LmWT parasites, both LmCen −/− immunized and healed mice generated a comparable CD4 + Ly6C + IFN-γ + effector T cells response 8 , which has been shown to play a role in parasite killing upon re-infection 30 .…”

Section: Discussionmentioning

confidence: 99%

“…Recently, using CRISPR gene editing, we have generated a centrin gene deleted live attenuated Leishmania major strain, LmCen -/- (8). We demonstrated that LmCen -/parasites do not cause lesions but have the ability to mount an immunological response that is protective against both cutaneous and visceral leishmaniasis in animal models that mimic human disease 8,9 .…”

Section: Introductionmentioning

confidence: 99%

Skin resident memory cells generated by the L. major centrin gene deleted parasites mediate protective immune response analogous to leishmanization

Ismail¹,

Karmakar²,

Bhattacharya³

et al. 2021

Preprint

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Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene deleted parasite strain (LmCen-/-) that induced protection against a homologous and heterologous challenges. The protection is mediated by IFN-gamma; secreting CD4+ T effector cells and multifunctional T cells, which is analogous to leishmanization. Previously, skin tissue resident memory T cells (TRM cells) were shown to be crucial for host protection in a leishmanization model. In this study, we evaluated generation and function of skin TRM cells following immunization with LmCen-/- parasites and compared those with leishmanization. In the absence of recoverable LmCen-/- parasites, the skin of immunized mice showed functional TRM cells comparable to leishmanized mice. The generation of the skin TRM cells was supported by the induction of cytokines and chemokines essential for their production and survival. Following challenge infection with wild type L. major, TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice which was similar to leishmanization. Further, upon challenge, CD4+ TRM cells induced higher levels of IFN-gamma; and Granzyme B in the immunized and leishmanized mice than non-immunized mice. Taken together, our studies demonstrate that a genetically modified live attenuated Leishmania vaccine generates functional CD4+ TRM cells that mediate protection and can be a safer alternative to leishmanization.

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Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis

Cited by 37 publications

References 49 publications

Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing

Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing

Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi

Skin resident memory cells generated by the L. major centrin gene deleted parasites mediate protective immune response analogous to leishmanization

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